bims-tunefa Biomed News
on Tumor necrosis factor superfamily and post-translational modifications
Issue of 2020‒09‒06
twenty-four papers selected by
John Silke
Walter and Eliza Hall Institute of Medical Research


  1. Biochem Pharmacol. 2020 Aug 28. pii: S0006-2952(20)30447-0. [Epub ahead of print] 114211
    Liang Y, Nandakumar KS, Cheng K.
      Proteolysis-targeting chimeras (PROTACs), the hetero-bifunctional compounds containing a specific ligand to bind the target protein, a suitable linker, and an E3 ubiquitin ligase substrate, are being developed for therapeutic applications. PROTACs hijack the catalytic activity of ubiquitin E3 ligases to mediate proteasome dependent degradation of selected protein of interest (POI), by bringing the ligase and POI into close spatial proximity and initiating the poly-ubiquitination process. Compared to the traditional small-molecule drugs, PROTACs reduce the problems of dosage, drug resistance, side effects and undruggable targets that could not be targeted pharmacologically. In this review, all the POIs, and peptide to small-molecule based PROTACs developed during the past two decades are summarized and directions for future development are discussed.
    Keywords:  Degradation; PROTACs; Small-molecule drug; Ubiquitination
    DOI:  https://doi.org/10.1016/j.bcp.2020.114211
  2. Nat Rev Drug Discov. 2020 Sep 03.
    Curtin NJ, Szabo C.
      The process of poly(ADP-ribosyl)ation and the major enzyme that catalyses this reaction, poly(ADP-ribose) polymerase 1 (PARP1), were discovered more than 50 years ago. Since then, advances in our understanding of the roles of PARP1 in cellular processes such as DNA repair, gene transcription and cell death have allowed the investigation of therapeutic PARP inhibition for a variety of diseases - particularly cancers in which defects in DNA repair pathways make tumour cells highly sensitive to the inhibition of PARP activity. Efforts to identify and evaluate potent PARP inhibitors have so far led to the regulatory approval of four PARP inhibitors for the treatment of several types of cancer, and PARP inhibitors have also shown therapeutic potential in treating non-oncological diseases. This Review provides a timeline of PARP biology and medicinal chemistry, summarizes the pathophysiological processes in which PARP plays a role and highlights key opportunities and challenges in the field, such as counteracting PARP inhibitor resistance during cancer therapy and repurposing PARP inhibitors for the treatment of non-oncological diseases.
    DOI:  https://doi.org/10.1038/s41573-020-0076-6
  3. FEBS J. 2020 Aug 31.
    LeBlanc N, Mallette E, Zhang W.
      Ubiquitination plays an essential role in signal transduction to regulate most if not all cellular processes. Among the enzymes that are involved in the ubiquitin (Ub) signaling cascade, tremendous efforts have been focused on elucidating the roles of E3 Ub ligases as they determine the complexity and specificity of ubiquitination. Not surprisingly, the malfunction of E3 ligases is directly implicated in many human diseases, including cancer. Therefore, there is an urgent need to develop potent and specific molecules to modulate E3 ligase activity as intracellular probes for target validation and as pharmacological agents in pre-clinical research. Unfortunately, the progress has been hampered by the dynamic regulation mechanisms for different types of E3 ligases. Here, we summarize the progress of using protein engineering to develop Ub variant (UbV) inhibitors for all major families of E3 ligases and UbV activators for HECT E3s and homodimeric RING E3s. We believe that this provides a general strategy and a valuable toolkit for the research community to inhibit or activate E3 ligases and these synthetic molecules have important implications in exploring protein degradation for drug discovery.
    Keywords:  Activator; Cancer; E3 ligases; Inhibitor; Phage display; Protein engineering; Signal transduction; Ubiquitin variant; Ubiquitination
    DOI:  https://doi.org/10.1111/febs.15536
  4. Open Biol. 2020 Sep;10(9): 200099
    Totzke J, Scarneo SA, Yang KW, Haystead TAJ.
      Aberrant tumour necrosis factor (TNF) signalling is a hallmark of many inflammatory diseases including rheumatoid arthritis (RA), irritable bowel disease and lupus. Maladaptive TNF signalling can lead to hyper active downstream nuclear factor (NF)-κβ signalling in turn amplifying a cell's inflammatory response and exacerbating disease. Within the TNF intracellular inflammatory signalling cascade, transforming growth factor-β-activated kinase 1 (TAK1) has been shown to play a critical role in mediating signal transduction and downstream NF-κβ activation. Owing to its role in TNF inflammatory signalling, TAK1 has become a potential therapeutic target for the treatment of inflammatory diseases such as RA. This review highlights the current development of targeting the TNF-TAK1 signalling axis as a novel therapeutic strategy for the treatment of inflammatory diseases.
    Keywords:  TAK1; TNF; inflammation; kinase; rheumatoid arthritis
    DOI:  https://doi.org/10.1098/rsob.200099
  5. Biochem Soc Trans. 2020 Sep 01. pii: BST20200225. [Epub ahead of print]
    Phan TK, Ozkocak DC, Poon IKH.
      Extracellular vesicles (EVs), membrane-bound vesicles that are naturally released by cells, have emerged as new therapeutic opportunities. EVs, particularly exosomes and microvesicles, can transfer effector molecules and elicit potent responses in recipient cells, making them attractive therapeutic targets and drug delivery platforms. Furthermore, containing predictive biomarkers and often being dysregulated in various disease settings, these EVs are being exploited for diagnostic purposes. In contrast, the therapeutic application of apoptotic bodies (ApoBDs), a distinct type of EVs released by cells undergoing a form of programmed cell death called apoptosis, has been largely unexplored. Recent studies have shed light on ApoBD biogenesis and functions, promisingly implicating their therapeutic potential. In this review, we discuss many strategies to develop ApoBD-based therapies as well as highlight their advantages and challenges, thereby positioning ApoBD for potential EV-based therapy.
    Keywords:  apoptosis; apoptotic bodies; extracellular vesicles; therapeutics
    DOI:  https://doi.org/10.1042/BST20200225
  6. Nat Chem. 2020 Aug 31.
    Brighty GJ, Botham RC, Li S, Nelson L, Mortenson DE, Li G, Morisseau C, Wang H, Hammock BD, Sharpless KB, Kelly JW.
      Drug candidates that form covalent linkages with their target proteins have been underexplored compared with the conventional counterparts that modulate biological function by reversibly binding to proteins, in part due to concerns about off-target reactivity. However, toxicity linked to off-target reactivity can be minimized by using latent electrophiles that only become activated towards covalent bond formation on binding a specific protein. Here we study sulfuramidimidoyl fluorides, a class of weak electrophiles that undergo sulfur(VI) fluoride exchange chemistry. We show that equilibrium binding of a sulfuramidimidoyl fluoride to a protein can allow nucleophilic attack by a specific amino acid side chain, which leads to conjugate formation. We incubated small molecules, each bearing a sulfuramidimidoyl fluoride electrophile, with human cell lysate, and the protein conjugates formed were identified by affinity chromatography-mass spectrometry. This inverse drug discovery approach identified a compound that covalently binds to and irreversibly inhibits the activity of poly(ADP-ribose) polymerase 1, an important anticancer target in living cells.
    DOI:  https://doi.org/10.1038/s41557-020-0530-4
  7. Nat Rev Mol Cell Biol. 2020 Sep 01.
    Bedoui S, Herold MJ, Strasser A.
      The removal of functionally dispensable, infected or potentially neoplastic cells is driven by programmed cell death (PCD) pathways, highlighting their important roles in homeostasis, host defence against pathogens, cancer and a range of other pathologies. Several types of PCD pathways have been described, including apoptosis, necroptosis and pyroptosis; they employ distinct molecular and cellular processes and differ in their outcomes, such as the capacity to trigger inflammatory responses. Recent genetic and biochemical studies have revealed remarkable flexibility in the use of these PCD pathways and indicate a considerable degree of plasticity in their molecular regulation; for example, despite having a primary role in inducing pyroptosis, inflammatory caspases can also induce apoptosis, and conversely, apoptotic stimuli can trigger pyroptosis. Intriguingly, this flexibility is most pronounced in cellular responses to infection, while apoptosis is the dominant cell death process through which organisms prevent the development of cancer. In this Review, we summarize the mechanisms of the different types of PCD and describe the physiological and pathological processes that engage crosstalk between these pathways, focusing on infections and cancer. We discuss the intriguing notion that the different types of PCD could be seen as a single, coordinated cell death system, in which the individual pathways are highly interconnected and can flexibly compensate for one another.
    DOI:  https://doi.org/10.1038/s41580-020-0270-8
  8. J Cell Mol Med. 2020 Sep 03.
    Zhong Y, Wu S, Yang Y, Li GQ, Meng L, Zheng QY, Li Y, Xu GL, Zhang KQ, Peng KF.
      Sepsis-associated acute kidney injury (SA-AKI) is a common clinical critical care syndrome. It has received increasing attention due to its high morbidity and mortality; however, its pathophysiological mechanisms remain elusive. LIGHT, the 14th member of the tumour necrosis factor (TNF) superfamily and a bidirectional immunoregulatory molecule that regulates inflammation, plays a pivotal role in disease pathogenesis. In this study, mice with an intraperitoneal injection of LPS and HK-2 cells challenged with LPS were employed as a model of SA-AKI in vivo and in vitro, respectively. LIGHT deficiency notably attenuated kidney injury in pathological damage and renal function and markedly mitigated the inflammatory reaction by decreasing inflammatory mediator production and inflammatory cell infiltration in vivo. The TLR4-Myd88-NF-κB signalling pathway in the kidney of LIGHT knockout mice was dramatically down-regulated compared to the controls. Recombinant human LIGHT aggravated LPS-treated HK-2 cell injury by up-regulating the expression of the TLR4-Myd88-NF-κB signalling pathway and inflammation levels. TAK 242 (a selective TLR4 inhibitor) reduced this trend to some extent. In addition, blocking LIGHT with soluble receptor fusion proteins HVEM-Fc or LTβR-Fc in mice attenuated renal dysfunction and pathological damage in SA-AKI. Our findings indicate that LIGHT aggravates inflammation and promotes kidney damage in LPS-induced SA-AKI via the TLR4-Myd88-NF-κB signalling pathway, which provide potential strategies for the treatment of SA-AKI.
    Keywords:  LIGHT; NF-κB; TLR4; acute kidney injury; sepsis
    DOI:  https://doi.org/10.1111/jcmm.15815
  9. Autophagy. 2020 Aug 31.
    Qiang L, Yang S, Cui YH, He YY.
      Macroautophagy/autophagy is a cellular catabolic process that is implicated in several physiological and pathological processes. However, the role of epidermal autophagy in wound healing remains unknown. Here, using mice with genetic ablation of the essential Atg5 (autophagy related 5) or Atg7 in their epidermis to inhibit autophagy, we show that keratinocyte autophagy regulates wound healing in mice. Wounding induces the expression of autophagy genes in mouse skin. Epidermis-specific autophagy deficiency inhibits wound closure, re-epithelialization, keratinocyte proliferation and differentiation, dermal granulation tissue formation, and infiltration of immune cells including macrophages, neutrophils, and mast cells, while it does not affect angiogenesis. Using cytokine array screening, we found that autophagy deficiency inhibits the transcription and production of the cytokine CCL2/MCP-1 by TNF. At the molecular level, TNF induces autophagic flux and the expression of autophagy genes through NFKB in epidermal keratinocytes. TNF promotes CCL2 transcription through the autophagy-AMPK-BRAF-MAPK/ERK-activator protein 1 (AP-1) pathway. Indeed, treating mice with recombinant CCL2 can reverse the effect of autophagy deficiency in keratinocytes. At the cellular level, we found that CCL2 induction via autophagy in keratinocytes is required not only for keratinocyte migration and proliferation but also for dermal fibroblast activation. Our findings demonstrate a critical role of epidermal autophagy in wound healing in vivo and elucidate a critical molecular machinery coordinating keratinocyte-fibroblast interaction in skin repair.
    Keywords:  CCL2/MCP-1; TNF; autophagy; differentiation; fibroblast; inflammation; keratinocyte; migration; proliferation; wound healing
    DOI:  https://doi.org/10.1080/15548627.2020.1816342
  10. J Exp Med. 2020 Dec 07. pii: e20191166. [Epub ahead of print]217(12):
    Oda SK, Anderson KG, Ravikumar P, Bonson P, Garcia NM, Jenkins CM, Zhuang S, Daman AW, Chiu EY, Bates BM, Greenberg PD.
      Adoptive T cell therapy (ACT) with genetically modified T cells has shown impressive results against some hematologic cancers, but efficacy in solid tumors can be limited by restrictive tumor microenvironments (TMEs). For example, Fas ligand is commonly overexpressed in TMEs and induces apoptosis in tumor-infiltrating, Fas receptor-positive lymphocytes. We engineered immunomodulatory fusion proteins (IFPs) to enhance ACT efficacy, combining an inhibitory receptor ectodomain with a costimulatory endodomain to convert negative into positive signals. We developed a Fas-4-1BB IFP that replaces the Fas intracellular tail with costimulatory 4-1BB. Fas-4-1BB IFP-engineered murine T cells exhibited increased pro-survival signaling, proliferation, antitumor function, and altered metabolism in vitro. In vivo, Fas-4-1BB ACT eradicated leukemia and significantly improved survival in the aggressive KPC pancreatic cancer model. Fas-4-1BB IFP expression also enhanced primary human T cell function in vitro. Thus, Fas-4-1BB IFP expression is a novel strategy to improve multiple T cell functions and enhance ACT against solid tumors and hematologic malignancies.
    DOI:  https://doi.org/10.1084/jem.20191166
  11. J Biol Chem. 2020 08 31. pii: jbc.REV120.012960. [Epub ahead of print]
    Cheloha RW, Harmand TJ, Wijne C, Schwartz TU, Ploegh HL.
      Reagents that bind tightly and specifically to biomolecules of interest remain essential in the exploration of biology and in their ultimate application to medicine. Besides ligands for receptors of known specificity, agents commonly used for this purpose are monoclonal antibodies derived from mice, rabbits, and other animals. However, such antibodies can be expensive to produce, challenging to engineer, and are not necessarily stable in the context of the cellular cytoplasm, a reducing environment. Heavy chain-only antibodies, discovered in camelids, have been truncated to yield single domain antibody fragments (VHHs or nanobodies) that overcome many of these shortcomings. While known as crystallization chaperones for membrane proteins or as simple alternatives to conventional antibodies, nanobodies have been applied in settings where the use of standard antibodies or their derivatives would be impractical or impossible. We review recent examples in which the unique properties of nanobodies have been combined with complementary methods, such as chemical functionalization, to provide tools with unique and useful properties.
    Keywords:  antibody engineering; cell signaling; protein chemistry; single-domain antibody (sdAb,nanobody); synthetic biology
    DOI:  https://doi.org/10.1074/jbc.REV120.012960
  12. Cell Mol Life Sci. 2020 Sep 01.
    Laham AJ, Saber-Ayad M, El-Awady R.
      Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a dual kinase that can phosphorylate its own activation loop on tyrosine residue and phosphorylate its substrates on threonine and serine residues. It is the most studied member of DYRK kinases, because its gene maps to human chromosome 21 within the Down syndrome critical region (DSCR). DYRK1A overexpression was found to be responsible for the phenotypic features observed in Down syndrome such as mental retardation, early onset neurodegenerative, and developmental heart defects. Besides its dual activity in phosphorylation, DYRK1A carries the characteristic of duality in tumorigenesis. Many studies indicate its possible role as a tumor suppressor gene; however, others prove its pro-oncogenic activity. In this review, we will focus on its multifaceted role in tumorigenesis by explaining its participation in some cancer hallmarks pathways such as proliferative signaling, transcription, stress, DNA damage repair, apoptosis, and angiogenesis, and finally, we will discuss targeting DYRK1A as a potential strategy for management of cancer and neurodegenerative disorders.
    Keywords:  53BP1; Alzheimer; Cancer therapy; Carcinogenesis; DNA damage; DNA repair; RNF169; p53
    DOI:  https://doi.org/10.1007/s00018-020-03626-4
  13. Expert Opin Biol Ther. 2020 Sep 04. 1-6
    Prignano F, Choi J, Pieper B, Iversen L.
      INTRODUCTION: Anti-tumor necrosis factor agents are key treatment options in moderate-severe psoriasis. The advent of multiple biosimilars of these drugs provides a major opportunity to address this particular factor by helping to reduce costs. Reduced cost can help improve undertreatment, which is one of the challenges in treating moderate-severe psoriasis. There is now a wealth of real-world evidence demonstrating that patients with psoriasis can be initiated on - or transitioned to - an anti-TNF biosimilar without detrimental effects on overall safety and efficacy. Furthermore, recent results suggest that patients can be switched between different biosimilar versions of the same anti-TNF agent without any compromise in outcomes.AREAS COVERED: In this review, we summarized the role of anti-TNFs in psoriasis, health economic aspects of anti-TNF biosimilars, and their real-world data in clinical practice and registries.
    EXPERT OPINION: The introduction and competition of anti-TNF biosimilars reduced the cost of biologics and accumulated real-world data support efficacy and safety of anti-TNF biosimilars for psoriasis treatment. Although IL-17 and IL-23 inhibitors show better efficacy in psoriasis patients, long-term efficacy and safety data of anti-TNF and cost-effectiveness of anti-TNF biosimilars may play an important role to increase patient access to biologics through greater adoption of biosimilars.
    Keywords:  Adalimumab; anti-TNF; biosimilar; etanercept; infliximab; psoriasis
    DOI:  https://doi.org/10.1080/14712598.2020.1812576
  14. FEBS Lett. 2020 Aug 31.
    Vasilenko EA, Gorshkova EN, Astrakhantseva IV, Drutskaya MS, Tillib SV, Nedospasov SA, Mokhonov VV.
      Spatial organization and conformational changes of antibodies may significantly affect their biological functions. We assessed the effect of mutual organization of the two VH H domains within bispecific antibodies recognizing human TNF and the surface molecules of murine myeloid cells (F4/80 or CD11b) on TNF retention and inhibition. TNF-neutralizing properties in vitro and in vivo of MYSTI-2 and MYSTI-3 antibodies were compared with new variants with interchanged VH H domains and different linker sequences. The most effective structure of MYSTI-2 and MYSTI-3 proteins required the Ser/Gly-containing 'superflexible' linker. The orientation of the modules was crucial for the activity of the proteins, but not for MYSTI-3 with the Pro/Gln-containing 'semi-rigid' linker. Our results may contribute toward the development of more effective drug prototypes.
    Keywords:  VHH; anti-TNF therapy; bispecific antibodies; linkers
    DOI:  https://doi.org/10.1002/1873-3468.13913
  15. Sci Rep. 2020 Aug 31. 10(1): 14267
    Traenkle B, Segan S, Fagbadebo FO, Kaiser PD, Rothbauer U.
      Epitope tagging is a versatile approach to study different proteins using a well-defined and established methodology. To date, most epitope tags such as myc, HA, V5 and FLAG tags are recognized by antibodies, which limits their use to fixed cells, tissues or protein samples. Here we introduce a broadly applicable tagging strategy utilizing a short peptide tag (PepTag) which is specifically recognized by a nanobody (PepNB). We demonstrated that the PepNB can be easily functionalized for immunoprecipitation or direct immunofluorescence staining of Pep-tagged proteins in vitro. For in cellulo studies we converted the PepNB into a fluorescently labeled Pep-chromobody (PepCB) which is functionally expressed in living cells. The addition of the small PepTag does not interfere with the examined structures in different cellular compartments and its detection with the PepCB enables optical antigen tracing in real time. By employing the phenomenon of antigen-mediated chromobody stabilization (AMCBS) using a turnover-accelerated PepCB we demonstrated that the system is suitable to visualize and quantify changes in Pep-tagged antigen concentration by quantitative live-cell imaging. We expect that this novel tagging strategy offers new opportunities to study the dynamic regulation of proteins, e.g. during cellular signaling, cell differentiation, or upon drug action.
    DOI:  https://doi.org/10.1038/s41598-020-71091-x
  16. Cell. 2020 Sep 03. pii: S0092-8674(20)30937-5. [Epub ahead of print]182(5): 1067-1071
    Morrison M, Merlo K, Woessner Z.
      We can maximize the impact of scientific conferences by uploading all conference presentations, posters, and abstracts to highly trafficked public repositories for each content type. Talks can be hosted on sites like YouTube and Youku, posters can be published on Figshare, and papers and abstracts can become open access preprints.
    DOI:  https://doi.org/10.1016/j.cell.2020.07.029
  17. J Clin Med. 2020 Aug 31. pii: E2821. [Epub ahead of print]9(9):
    Sisto M, Ribatti D, Lisi S.
      Sjögren's syndrome (SS) is a systemic autoimmune inflammatory disease with a poorly defined aetiology, which targets exocrine glands (particularly salivary and lachrymal glands), affecting the secretory function. Patients suffering from SS exhibit persistent xerostomia and keratoconjunctivitis sicca. It is now widely acknowledged that a chronic grade of inflammation plays a central role in the initiation, progression, and development of SS. Consistent with its key role in organizing inflammatory responses, numerous recent studies have shown involvement of the transcription factor nuclear factor κ (kappa)-light-chain-enhancer of activated B cells (NF-κB) in the development of this disease. Therefore, chronic inflammation is considered as a critical factor in the disease aetiology, offering hope for the development of new drugs for treatment. The purpose of this review is to describe the current knowledge about the NF-κB-mediated molecular events implicated in the pathogenesis of SS.
    Keywords:  NF-κB; Sjögren’s syndrome; inflammation
    DOI:  https://doi.org/10.3390/jcm9092821
  18. F1000Res. 2020 ;pii: F1000 Faculty Rev-1013. [Epub ahead of print]9
    Rose-John S.
      Biochemically, interleukin-6 belongs to the class of four-helical cytokines. The cytokine can be synthesised and secreted by many cells. It acts via a cell surface-expressed interleukin-6 receptor, which is not signalling competent. This receptor, when complexed with interleukin-6, associates with the signalling receptor glycoprotein 130 kDa (gp130), which becomes dimerised and initiates intracellular signalling via the Janus kinase/signal transducer and activator of transcription and rat sarcoma proto oncogene/mitogen-activated protein kinase/phosphoinositide-3 kinase pathways. Physiologically, interleukin-6 is involved in the regulation of haematopoiesis and the coordination of the innate and acquired immune systems. Additionally, interleukin-6 plays an important role in the regulation of metabolism, in neural development and survival, and in the development and maintenance of various cancers. Although interleukin-6 is mostly regarded as a pro-inflammatory cytokine, there are numerous examples of protective and regenerative functions of this cytokine. This review will explain the molecular mechanisms of the, in part opposing, activities of the cytokine interleukin-6.
    Keywords:  ADAM17; IL-6; IL-6R; gp130; sIL-6R; sgp130Fc; trans-signalling
    DOI:  https://doi.org/10.12688/f1000research.26058.1
  19. Nature. 2020 Sep 01.
    Clements JC.
      
    Keywords:  Careers; Ethics; Publishing
    DOI:  https://doi.org/10.1038/d41586-020-02512-0
  20. Cell Stem Cell. 2020 Sep 03. pii: S1934-5909(20)30403-3. [Epub ahead of print]27(3): 349-351
    Ramos R, Plikus MV.
      Large skin wounds in adult mice can heal by regenerating new hair follicles and adipocytes in their center. In this issue of Cell Stem Cell, Abbasi et al. (2020) report how typically scar-forming wound fibroblasts enter a transcriptionally distinct single-cell state of competence during regeneration of new hair follicle mesenchyme.
    DOI:  https://doi.org/10.1016/j.stem.2020.08.007
  21. Nat Rev Drug Discov. 2020 Sep 02.
    Attwood MM, Jonsson J, Rask-Andersen M, Schiöth HB.
      Historically, the main classes of drug targets have been receptors, enzymes, ion channels and transporters. However, owing largely to the rise of antibody-based therapies in the past two decades, soluble protein ligands such as inflammatory cytokines have become an increasingly important class of drug targets. In this Review, we analyse drugs targeting ligands that have reached clinical development at some point since 1992. We identify 291 drugs that target 99 unique ligands, and we discuss trends in the characteristics of the ligands, drugs and indications for which they have been tested. In the last 5 years, the number of ligand-targeting drugs approved by the FDA has doubled to 34, while the number of clinically validated ligand targets has doubled to 22. Cytokines and growth factors are the predominant types of targeted ligands (70%), and inflammation and autoimmune disorders, cancer and ophthalmological diseases are the top therapeutic areas for both approved agents and agents in clinical studies, reflecting the central role of cytokine and/or growth factor pathways in such diseases.
    DOI:  https://doi.org/10.1038/s41573-020-0078-4
  22. ACS Cent Sci. 2020 Aug 26. 6(8): 1376-1385
    Straková K, López-Andarias J, Jiménez-Rojo N, Chambers JE, Marciniak SJ, Riezman H, Sakai N, Matile S.
      Tools to image membrane tension in response to mechanical stimuli are badly needed in mechanobiology. We have recently introduced mechanosensitive flipper probes to report quantitatively global membrane tension changes in fluorescence lifetime imaging microscopy (FLIM) images of living cells. However, to address specific questions on physical forces in biology, the probes need to be localized precisely in the membrane of interest (MOI). Herein we present a general strategy to image the tension of the MOI by tagging our newly introduced HaloFlippers to self-labeling HaloTags fused to proteins in this membrane. The critical challenge in the construction of operational HaloFlippers is the tether linking the flipper and the HaloTag: It must be neither too taut nor too loose, be hydrophilic but lipophilic enough to passively diffuse across membranes to reach the HaloTags, and allow partitioning of flippers into the MOI after the reaction. HaloFlippers with the best tether show localized and selective fluorescence after reacting with HaloTags that are close enough to the MOI but remain nonemissive if the MOI cannot be reached. Their fluorescence lifetime in FLIM images varies depending on the nature of the MOI and responds to myriocin-mediated sphingomyelin depletion as well as to osmotic stress. The response to changes in such precisely localized membrane tension follows the validated principles, thus confirming intact mechanosensitivity. Examples covered include HaloTags in the Golgi apparatus, peroxisomes, endolysosomes, and the ER, all thus becoming accessible to the selective fluorescence imaging of membrane tension.
    DOI:  https://doi.org/10.1021/acscentsci.0c00666